Bifidobacteria for treating diabetes and related conditions

ABSTRACT

This invention relates to new uses of  Bifidobacteria  (particularly, although not exclusively, probiotic  Bifidobacteria ), and to food products, feed products, dietary supplements and pharmaceutical formulations containing them. The bacteria are suitable for the treatment of diabetes (particularly Type 2 diabetes), obesity and related conditions, metabolic syndrome, insulin resistance, and impaired glucose metabolism and consequences thereof, lowering tissue inflammation, treating hepatitis, myositis and cardiovascular conditions.

FIELD OF THE INVENTION

This invention relates to new uses of Bifidobacteria (particularly,although not exclusively, probiotic Bifidobacteria), and to foodproducts, feed products, dietary supplements and pharmaceuticalformulations containing them.

DESCRIPTION OF THE PRIOR ART

Diabetes mellitus, often referred to simply as diabetes, is a conditioncharacterized by disordered metabolism and abnormally high blood sugar(hyperglycaemia) resulting from insufficient levels and/or action of thehormone insulin. The characteristic symptoms are excessive urineproduction (polyuria) due to high blood glucose levels, excessive thirstand increased fluid intake (polydipsia) attempting to compensate forincreased urination, blurred vision due to high blood glucose effects onthe eye's optics, unexplained weight loss, and lethargy. These symptomsare likely to be less apparent if the blood sugar is only mildlyelevated.

The World Health Organisation recognises three main forms of diabetesmellitus: type 1, type 2, and gestational diabetes (occurring duringpregnancy), which have different causes and population distributions.While, ultimately, all forms are due to the beta cells of the pancreasbeing unable to produce sufficient insulin to prevent hyperglycemia, thecauses are different. Type 1 diabetes is usually due to autoimmunedestruction of the pancreatic beta cells. Type 2 diabetes ischaracterized by insulin resistance in target tissues. This causes aneed for abnormally high amounts of insulin and diabetes develops whenthe beta cells cannot meet this demand. Gestational diabetes is similarto type 2 diabetes in that it involves insulin resistance; the hormonesof pregnancy can cause insulin resistance in women geneticallypredisposed to developing this condition.

Gestational diabetes typically resolves with delivery of the child:however, types 1 and 2 diabetes are chronic conditions. All types havebeen treatable since insulin became medically available in 1921. Type 1diabetes, in which insulin is not secreted by the pancreas, is directlytreatable only with injected insulin, although dietary and otherlifestyle adjustments are part of management. Type 2 may be managed witha combination of dietary treatment, tablets and injections and,frequently, insulin supplementation.

Diabetes can cause many complications. Acute complications(hypoglycemia, ketoacidosis or nonketotic hyperosmolar coma) may occurif the disease is not adequately controlled. Serious long-termcomplications include cardiovascular disease (doubled risk), chronicrenal failure, retinal damage (which can lead to blindness), nervedamage (of several kinds), and microvascular damage, which may causeimpotence and poor healing. Poor healing of wounds, particularly of thefeet, can lead to gangrene, which may require amputation. Adequatetreatment of diabetes, as well as increased emphasis on blood pressurecontrol and lifestyle factors (such as not smoking and keeping a healthybody weight), may improve the risk profile of most aforementionedcomplications. In the developed world, diabetes is the most significantcause of adult blindness in the non-elderly and the leading cause ofnon-traumatic amputation in adults, and diabetic nephropathy is the mainillness requiring renal dialysis in the United States.

Diabetes mellitus is currently a chronic disease, without a cure, andmedical emphasis must necessarily be on managing/avoiding possibleshort-term as well as long-term diabetes-related problems. There is anexceptionally important role for patient education, dietetic support,sensible exercise, self glucose monitoring, with the goal of keepingboth short-term blood glucose levels, and long term levels as well,within acceptable bounds. Careful control is needed to reduce the riskof long term complications. This is theoretically achievable withcombinations of diet, exercise and weight loss (type 2), various oraldiabetic drugs (type 2 only), and insulin use (type 1 and increasinglyfor type 2 not responding to oral medications). In addition, given theassociated higher risks of cardiovascular disease, lifestylemodifications should be undertaken to control blood pressure andcholesterol by exercising more, smoking cessation, consuming anappropriate diet, wearing diabetic socks, and if necessary, taking anyof several drugs to reduce pressure.

Oral antidiabetic drugs and insulin analogs currently on the market orundergoing clinical trials include biguanides (such as metformin),sulfonylureas (such as carbutamide, chlorpropamide, glibenclamide(Glyburide), gliclazide, glimepiride, glipizide, gliquidone, tolazamideor tolbutamide), alpha-glucosidase inhibitors (such as acarbose,miglitol or voglibose), thiazolidinediones (TZD) (such as pioglitazone,rivoglitazone or rosiglitazone), meglitinides (such as nateglinide,repaglinide or mitiglinide), dipeptidyl peptidase-4 (DPP-4) inhibitors(such as alogliptin, saxagliptin, sitagliptin car vildagliptin),glucagon-like peptide-1 analogs (such as exenatide, liraglutide, oralbiglutide), amylin analogs (such as pramlintide), fast acting insulinanalogs (such as insulin lispro, insulin aspart and insulin glulisine),long acting insulin analogs (such as insulin glargine, insulin detemir),dual PPAR agonists (such as aleglitazar) and SGLT2 inhibitors (such asdapagliflozin, remogliflozin and sergliflozin).

Type 2 diabetes is often associated with obesity. The body mass index(BMI) (calculated as weight in kilograms divided by the square of heightin metres) is the most commonly accepted measurement for overweightand/or obesity. A BMI exceeding 25 is considered overweight. Obesity isdefined as a BMI of 30 or more, with a BMI of 35 or more considered asserious comorbidity obesity and a BMI of 40 or more considered morbidobesity. Mortality is increased in obesity, with a BMI of over 32 beingassociated with a doubled risk of death. There are alterations in thebody's response to insulin (insulin resistance), a proinflammatory stateand an increased tendency to thrombosis (prothrombotic state).

Central obesity (male-type or waist-predominant obesity, characterisedby a high waist-hip ratio), is a particularly important risk factor fordiabetes and metabolic syndrome, the clustering of a number of diseasesand risk factors that heavily predispose for cardiovascular disease.These are diabetes mellitus type 2, high blood pressure, high bloodcholesterol, and triglyceride levels (combined hyperlipidemia).

The use of microorganisms in treating obesity, diabetes anddiabetes-related conditions is in general known in the art. For example,WO 2007/043933 describes the use of probiotic bacteria for themanufacture of food and feed products, dietary supplements, forcontrolling weight gain, preventing obesity, increasing satiety,prolonging satiation, reducing food intake, reducing fat deposition,improving energy metabolism, enhancing insulin sensitivity, treatingobesity and treating insulin insensitivity.

WO 2009/024429 describes the use of a primary composition comprising anagent that reduces the amount of proteobacteria, in particularenterobacteria and/or deferribacteres in the gut for the treatment orprevention of metabolic disorders, to support and/or to support weightmanagement.

WO 2009/004076 describes the use of probiotic bacteria for normalisingplasma glucose concentrations, improving insulin sensitivity, andreducing the risk of development in pregnant women, and preventinggestational diabetes.

WO 2009/021824 describes the use of probiotic bacteria, in particularLactobacillus rhamnosus, to treat obesity, treat metabolic disorders,and support weight loss and/or weight maintenance.

WO 2008/016214 describes a probiotic lactic acid bacterium of the strainLactobacillus gasseri BNR17 and its use in the inhibition of weightgain.

WO 02/38165 describes use of a strain of Lactobacillus (in particular,Lactobacillus plantarum) in reducing the risk factors involved in themetabolic syndrome.

US 2002/0037577 describes the use of microorganisms, such asLactobacilli, for the treatment or prevention of obesity or diabetesmellitus by reduction of the amount of monosaccharide or disaccharidewhich may be absorbed into the body, by converting such compounds intopolymeric materials which cannot be absorbed by the intestine.

Lee et al., J. Appl. Microbiol. 2007, 103, 1140-1146, describes theanti-obesity activity of trans-10, cis-12-conjugated linoleic acid(CLA)-producing bacterium of the strain Lactobacillus plantarum PL62 inmice.

Li et al., Hepatology, 2003, 37(2), 343-350, describe the use ofprobiotics and anti-TNF antibodies in a mouse model for non-alcoholicfatty liver disease.

SUMMARY OF THE INVENTION

in one aspect, the invention comprises use of a bacterium of the genusBifidobacterium or a mixture thereof in the manufacture of a foodproduct, dietary supplement or medicament for treating diabetes(preferably but not exclusively Type 2 diabetes) in a mammal.

In another aspect, the invention comprises use of a bacterium of thegenus Bifidobacterium or a mixture thereof in the manufacture of a foodproduct, dietary supplement or medicament for treating impaired glucosetolerance in a mammal.

In a further aspect, the invention comprises use of a bacterium of thegenus Bifidobacterium or a mixture thereof in the manufacture of a foodproduct, dietary supplement or medicament for normalising insulinsensitivity in a mammal.

In a yet further aspect, the invention comprises use of a bacterium ofthe genus Bifidobacterium or a mixture thereof in the manufacture of afood product, dietary supplement or medicament for increasing fedinsulin secretion in a mammal.

In a still further aspect, the invention comprises use of a bacterium ofthe genus Bifidobacterium or a mixture thereof in the manufacture of afood product, dietary supplement or medicament for decreasing fastedinsulin secretion in a mammal.

In an additional aspect, the invention comprises use of a bacterium ofthe genus Bifidobacterium or a mixture thereof in the manufacture of afood product, dietary supplement or medicament for improving glucosetolerance in a mammal.

In another aspect, the invention comprises use of a bacterium of thegenus Bifidobacterium or a mixture thereof in the manufacture of a foodproduct, dietary supplement or medicament for treating obesity,controlling weight gain and/or inducing weight loss in a mammal.

In a further aspect, the invention comprises use of a bacterium of thegenus Bifidobacterium or a mixture thereof in the manufacture of a foodproduct, dietary supplement or medicament for lowering body fat mass ina mammal.

In a yet further aspect, the invention comprises use of a bacterium ofthe genus Bifidobacterium or a mixture thereof in the manufacture of afood product, dietary supplement or medicament for lowering mesentericfat mass in a mammal.

In a still further aspect, the invention comprises use of a bacterium ofthe genus Bifidobacterium or a mixture thereof in the manufacture of afood product, dietary supplement or medicament for lowering tissueinflammation (particularly, although not exclusively, muscle tissueinflammation, liver tissue inflammation and/or adipose tissueinflammation) in a mammal.

In a still further aspect, the invention comprises use of a bacterium ofthe genus Bifidobacterium or a mixture thereof in the manufacture of afood product, dietary supplement or medicament for treating hepatitis ina mammal.

In a yet further aspect, the invention comprises use of a bacterium ofthe genus Bifidobacterium or a mixture thereof in the manufacture of afood product, dietary supplement or medicament for treating myositis ina mammal.

In a still further aspect, the invention comprises use of a bacterium ofthe genus Bifidobacterium or a mixture thereof in the manufacture of afood product, dietary supplement or medicament for treatingcardiovascular disease in a mammal.

In a yet further aspect, the invention comprises use of a bacterium ofthe genus Bifidobacterium or a mixture thereof in the manufacture of afood product, dietary supplement or medicament for treating metabolicsyndrome in a mammal.

In another aspect, the invention comprises a bacterium of the genusBifidobacterium or a mixture thereof for use in treating diabetes(particularly, although not exclusively, Type 2 diabetes) in a mammal.

In a further aspect, the invention comprises a bacterium of the genusBifidobacterium or a mixture thereof for use in treating impairedglucose tolerance in a mammal.

In a yet further aspect, the invention comprises a bacterium of thegenus Bifidobacterium or a mixture thereof for use in normalisinginsulin sensitivity in a mammal.

In a still further aspect, the invention comprises a bacterium of thegenus Bifidobacterium or a mixture thereof for use in increasing fedinsulin secretion in a mammal.

In another aspect, the invention comprises a bacterium of the genusBifidobacterium or a mixture thereof for use in decreasing fastedinsulin secretion in a mammal.

In a further aspect, the invention comprises a bacterium of the genusBifidobacterium or a mixture thereof for use in improving glucosetolerance in a mammal.

In a yet further aspect, the invention comprises a bacterium of thegenus Bifidobacterium or a mixture thereof for use in treating obesity,controlling weight gain and/or inducing weight loss in a mammal.

In another aspect, the invention comprises a bacterium of the genusBifidobacterium or a mixture thereof for use in lowering body fat massin a mammal.

In a further aspect, the invention comprises a bacterium of the genusBifidobacterium or a mixture thereof for use in lowering mesenteric fatmass in a mammal.

In a yet further aspect, the invention comprises a bacterium of thegenus Bifidobacterium or a mixture thereof for use in lowering tissueinflammation (particularly, although not exclusively, muscle tissueinflammation, liver tissue inflammation and/or adipose tissueinflammation) in a mammal.

In a still further aspect, the invention comprises a bacterium of thegenus Bifidobacterium or a mixture thereof for use in treating hepatitisin a mammal.

In a yet further aspect, the invention comprises a bacterium of thegenus Bifidobacterium or a mixture thereof for use in treating myositisin a mammal.

In a still further aspect, the invention comprises a bacterium of thegenus Bifidobacterium or a mixture thereof for use in treatingcardiovascular disease in a mammal.

In a still further aspect, the invention comprises a bacterium of thegenus Bifidobacterium or a mixture thereof for use in treating metabolicsyndrome in a mammal.

In another aspect, the invention comprises a method of treating diabetes(particularly although not exclusively Type 2 diabetes) in a mammal,comprising administering to a mammal in need of such treatment abacterium of the genus Bifidobacterium or a mixture thereof.

In a further aspect, the invention comprises a method of treatingimpaired glucose tolerance in a mammal, comprising administering to amammal in need thereof a bacterium of the genus Bifidobacterium or amixture thereof.

In a yet further aspect, the invention comprises a method of normalisinginsulin sensitivity in a mammal, comprising administering to a mammal inneed thereof a bacterium of the genus Bifidobacterium or a mixturethereof.

In a still further aspect, the invention comprises a method ofincreasing fed insulin secretion in a mammal, comprising administeringto a mammal in need thereof a bacterium of the genus Bifidobacterium ora mixture thereof.

In another aspect, the invention comprises a method of decreasing fastedinsulin secretion in a mammal, comprising administering to a mammal inneed thereof a bacterium of the genus Bifidobacterium or a mixturethereof.

In a further aspect, the invention comprises a method of improvingglucose tolerance in a mammal, comprising administering to a mammal inneed thereof a bacterium of the genus Bifidobacterium or a mixturethereof.

In a yet further aspect, the invention comprises a method of treatingobesity, controlling weight gain and/or inducing weight loss in amammal, comprising administering to a mammal in need of such treatment abacterium of the genus Bifidobacterium or a mixture thereof.

In a still further aspect, the invention comprises a method of loweringbody fat mass in a mammal, comprising administering to a mammal in needthereof a bacterium of the genus Bifidobacterium or a mixture thereof.

In another aspect, the invention comprises a method of loweringmesenteric fat mass in a mammal, comprising administering to a mammal inneed thereof a bacterium of the genus Bifidobacterium or a mixturethereof.

In a further aspect, the invention comprises a method of lowering tissueinflammation (particularly, although not exclusively, muscle tissueinflammation, liver tissue inflammation and/or adipose tissueinflammation) in a mammal, comprising administering to a mammal in needthereof a bacterium of the genus Bifidobacterium or a mixture thereof.

In a yet further aspect, the invention comprises a method of treatinghepatitis in a mammal, comprising administering to a mammal in needthereof a bacterium of the genus Bifidobacterium or a mixture thereof.

In a still further aspect, the invention comprises a method of treatingmyositis in a mammal, comprising administering to a mammal in needthereof a bacterium of the genus Bifidobacterium or a mixture thereof.

In a yet further aspect, the invention comprises a method of treatingcardiovascular disease in a mammal, comprising administering to a mammalin need thereof a bacterium of the genus Bifidobacterium or a mixturethereof.

In a still further aspect, the invention comprises a method of treatingmetabolic syndrome in a mammal, comprising administering to a mammal inneed of such treatment a bacterium of the genus Bifidobacterium or amixture thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the results of an intraperitoneal glucose tolerancetest in adult male C57bl6 mice fed a high fat diet (HFD) for four weeksor a normal chow (NC);

FIG. 2 illustrates the results of an intraperitoneal glucose tolerancetest four weeks after the beginning of the probiotic treatment in highfat diet fed mice;

FIG. 3 illustrates the effect of treatment with Bifidobacterium animalissubsp. lactis strain 420 (B420) Lactobacillus acidophilus strain NCFM(NCFM) or a combination of the two (B420+NCFM) on the body fat mass ofhigh fat diet fed mice;

FIG. 4 illustrates the effect of treatment with B420, NCFM or B420+NCFMon the free water weight of high fat diet fed mice;

FIG. 5 illustrates the effect of treatment with B420, NCFM or 420+NCFMon the lean body mass weight of high fat diet fed mice;

FIG. 6 illustrates the effect of treatment with B420, NCFM or B420+NCFMon the total water weight of high fat diet fed mice;

FIG. 7 illustrates the effect of treatment with B420, NCFM or B420+NCFMon the subcutaneous adipose tissue weight of high fat diet fed mice;

FIG. 8 illustrates the effect of treatment with B420, NCFM or B420+NCFMon the mesenteric adipose tissue weight of high fat diet fed mice;

FIG. 9 illustrates the effect of treatment with B420, NCFM or B420+NCFMon the liver weight of high fat diet fed mice;

FIG. 10 illustrates the body weight gain before and after treatment withB420, NCFM or B420+NCFM of high fat diet fed mice;

FIG. 11 illustrates the effect of treatment with B420, NCFM or B420+NCFMon the fasted and fed insulin levels of high fat diet fed mice;

FIG. 12 illustrates the effect of treatment with B420, NCFM or B420+NCFMon the insulin sensitivity of high fat diet fed mice;

FIG. 13 illustrates the liver cytokine mRNA concentrations in HFDdiabetic mice treated with B420, NCFM or B420+NCFM and control HFD-fedmice;

FIG. 14 illustrates the liver tissue inflammatory index in HFD diabeticmice treated with B420, NCFM or B420+NCFM and control HFD-fed mice;

FIG. 15 illustrates the skeletal muscle cytokine mRNA concentrations inHFD diabetic mice treated with B420, NCFM or B420+NCFM and controlHFD-fed mice;

FIG. 16 illustrates the inflammatory index of skeletal muscle tissues inHFD diabetic mice treated with B420, NCFM or B420+NCFM and controlHFD-fed mice.

FIG. 17 illustrates the subcutaneous adipose tissue cytokine mRNAconcentrations in HFD diabetic mice treated with B420, NCFM or B420+NCFand control HFD-fed mice;

FIG. 18 illustrates the inflammatory index of subcutaneous adiposetissues in HFD diabetic mice treated with B420, NCFM or B420+NCFM andcontrol HFD-fed mice;

FIG. 19 illustrates the blood glucose levels of HFD-fed mice treatedwith B420, a combination of B420 with polydextrose (B420+PDX), metformin(MET) or a combination of polydextrose and metformin (PDX+MET) andcontrol HFD-treated mice;

FIG. 20 illustrates the plasma insulin levels of HFD-fed mice treatedwith B420, B420+PDX, MET or a combination of B420 and metformin(B420+MET) and control HFD-treated mice; and

FIG. 21 illustrates the HOMA-IR (homeostatic model of insulinresistance) levels of HFD-fed mice treated with B420, B420+PDX, MET,B420+MET, PDX+MET and control HFD-treated mice.

DETAILED DESCRIPTION OF THE INVENTION

Bacteria

The bacterium used in the present invention is selected from aBifidobacterium or a mixture thereof. Preferably the Bifidobacterium tobe used in the present invention is a Bifidobacterium which is generallyrecognised as safe and, which is preferably GRAS approved.

The bacterium may be used in any form capable of exerting the effectsdescribed herein. For example, the bacteria may be viable, dormant,inactivated or dead bacteria. Preferably, the bacteria are viablebacteria.

The bacteria may comprise whole bacteria or may comprise bacterialcomponents. Examples of such components include bacterial cell wallcomponents such as peptidoglycan, bacterial nucleic acids such as DNAand RNA, bacterial membrane components, and bacterial structuralcomponents such as proteins, carbohydrates, lipids and combinations ofthese such as lipoproteins, glycolipids and glycoproteins.

The bacteria may also or alternatively comprise bacterial metabolites.In this specification the term ‘bacterial metabolites’ includes allmolecules produced or modified by the (probiotic) bacteria as a resultof bacterial metabolism during growth, survival, persistence, transit orexistence of bacteria during probiotic product manufacture and storageand during gastrointestinal transit in a mammal. Examples include allorganic acids, inorganic acids, bases, proteins and peptides, enzymesand co-enzymes, amino acids and nucleic acids, carbohydrates, lipids,glycoproteins, lipoproteins, glycolipids, vitamins, all bioactivecompounds, metabolites containing an inorganic component, and all smallmolecules, for example nitrous molecules or molecules containing asulphurous acid.

Preferably the bacteria comprise whole bacteria, more preferably wholeviable bacteria.

Preferably, the Bifidobacterium used in accordance with the presentinvention is one which is suitable for human and/or animal consumption.A skilled person will be readily aware of specific species and orstrains of Bifidobacteria from within the genera described herein whichare used in the food and/or agricultural industries and which aregenerally considered suitable for human and/or animal consumption.

In the present invention, the Bifidobacterium used may be of the sametype (species and strain) or may comprise a mixture of species and/orstrains.

Suitable Bifidobacteria are selected from the species Bifidobacteriumlactis, Bifidobacterium bifidium, Bifidobacterium longum,Bifidobacterium animalis, Bifidobacterium breve, Bifidobacteriuminfantis, Bifidobacterium catenulatum, Bifidobacteriumpseudocatenulatum, Bifidobacterium adolescentis, and Bifidobacteriumangulatum, and combinations of any thereof.

Preferably, the Bifidobacterium used in the present invention is of thespecies Bifidobacterium animalis. More preferably, the Bifidobacteriumused in the present invention is of the species Bifidobacterium animalissubsp. lactis.

In a particularly preferred embodiment, the bacteria used in the presentinvention are Bifidobacterium animalis subsp. lactis strain 420 (B420).This strain is commercially available from Danisco A/S.

In one embodiment, the bacterium used in the present invention is aprobiotic bacterium. In this specification the term ‘probioticbacterium’ is defined as covering any non-pathogenic bacterium which,when administered live in adequate amounts, confer a health benefit onthe host. These probiotic strains generally have the ability to survivethe passage through the upper part of the digestive tract. They arenon-pathogenic, non-toxic and exercise their beneficial effect on healthon the one hand via ecological interactions with the resident flora inthe digestive tract, and on the other hand via their ability toinfluence the immune system in a positive manner via the “GALT”(gut-associated lymphoid tissue). Depending on the definition ofprobiotics, these bacteria, when given in a sufficient number, have theability to progress live through the intestine, however they do notcross the intestinal barrier and their primary effects are thereforeinduced in the lumen and/or the wall of the gastrointestinal tract. Theythen form part of the resident flora during the administration period.This colonization (or transient colonization) allows the probioticbacteria to exercise a beneficial effect, such as the repression ofpotentially pathogenic micro-organisms present in the flora andinteractions with the immune system of the intestine.

In preferred embodiments, the bacterium used in the present invention isa probiotic Bifidobacterium.

In some embodiments, the Bifidobacterium is used in the presentinvention together with a bacterium of the genus Lactobacillus. Acombination of Bifidobacterium and Lactobacillus bacteria according tothe present invention exhibits a synergistic effect in certainapplications (i.e. an effect which is greater than the additive effectof the bacteria when used separately). For example, combinations which,in addition to having effect on the mammal as single components, mayhave beneficial effect on the other components of the combination, forexample by producing metabolites which are then in turn used as anenergy source by other components of the combination, or maintainingphysiological conditions which favour the other components.

Typically, the Lactobacillus bacteria are selected from the speciesLactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefiri,Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus helveticus,Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillussalivarius, Lactobacillus curvatus, Lactobacillus bulgaricus,Lactobacillus sakei, Lactobacillus reuteri, Lactobacillus fermentum,Lactobacillus farciminis, Lactobacillus lactis, Lactobacillusdelbreuckii, Lactobacillus plantarum, Lactobacillus paraplantarum,Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsoniiand Lactobacillus jensenii, and combinations of any thereof.

In preferred embodiments, the Lactobacillus bacterium used in thepresent invention is a probiotic Lactobacillus.

Preferably, the Lactobacillus bacterium used in the present invention ofthe species Lactobacillus acidophilus.

In a preferred embodiment, the Bifidobacterium is used in the presentinvention together with a bacterium of the species Lactobacillusacidophilus strain NCFM. Lactobacillus acidophilus NCFM was deposited byRhodia Chimie, France, at the American Type Culture Collection asPTA-4797 on 15 Nov. 2002.

In a particularly preferred embodiment, the bacteria used in the presentinvention comprise a combination of Bifidobacterium animalis subsp.lactis strain 420 (B420) and Lactobacillus acidophilus strain NCFM(PTA-4797).

Dosage

The Bifidobacterium (such as a strain of Bifidobacterium animalls subsp.lactis, for example Bifidobacterium animalis subsp. lactis strain 420(B420), and (if present) the Lactobacillus (such as a strain ofLactobacillus acidophilus, for example Lactobacillus acidophilus strainNCFM) used in accordance with the present invention may comprise from10⁶ to 10¹² CFU of bacteria/g of support, and more particularly from 10⁸to 10¹² CFU of bacteria/g of support, preferably 10⁹ to 10¹² CFU/g forthe lyophilized form.

Suitably, the Bifidobacterium (such as a strain of Bifidobacteriumanimalis subsp. lactis, for example Bifidobacterium animalis subsp.lactis strain 420 (B420), and (if present) the Lactobacillus (such as astrain of Lactobacillus acidophilus, for example Lactobacillusacidophilus strain NCFM), may be administered at a dosage of from about10⁶ to about 10¹² CFU of microorganism/dose, preferably about 10⁸ toabout 10¹² CFU of microorganism/dose. By the term “per dose” it is meantthat this amount of microorganism is provided to a subject either perday or per intake, preferably per day. For example, if the microorganismis to be administered in a food product (for example in a yoghurt)—thenthe yoghurt will preferably contain from about 10⁸ to 10¹² CFU of themicroorganism. Alternatively, however, this amount of microorganism maybe split into multiple administrations each consisting of a smalleramount of microbial loading—so long as the overall amount ofmicroorganism received by the subject in any specific time (for instanceeach 24 hour period) is from about 10⁶ to about 10¹² CFU ofmicroorganism, preferably 10⁸ to about 10¹² CFU of microorganism.

In accordance with the present invention an effective amount of at leastone strain of a microorganism may be at least 10⁶ CFU ofmicroorganism/dose, preferably from about 10⁶ to about 10¹² CFU ofmicroorganism/dose, preferably about 10⁸ to about 10¹² CFU ofmicroorganism/dose.

In one embodiment, preferably the Bifidobacterium (such as a strain ofBifidobacterium animalis subsp. lactis, for example Bifidobacteriumanimalis subsp. lactis strain 420 (B420), and (if present) theLactobacillus (such as a strain of Lactobacillus acidophilus, forexample Lactobacillus acidophilus strain NCFM), may be administered at adosage of from about 10⁶ to about 10¹² CFU of microorganism/day,preferably about 10⁸ to about 10¹² CFU of microorganism/day. Hence, theeffective amount in this embodiment may be from about 10⁶ to about 10¹²CFU of microorganism/day, preferably about 10⁸ to about 10¹² CFU ofmicroorganism/day.

CFU stands for “colony-forming units”. By ‘support’ is meant the foodproduct, dietary supplement or the pharmaceutically acceptable support.

When Bifidobacteria are used in the present invention together withLactobacilli, the bacteria may be present in any ratio capable ofachieving the desired effects of the invention described herein.Typically, the Bifidobacteria to Lactobacilli ratio (measured in termsof colony forming units) is in the range 1:100 to 100:1, suitably 1:50to 50:1, preferably 1:20 to 20:1, more preferably 1:10 to 10:1, stillmore preferably 1:5 to 5:1, yet more preferably 1:3 to 3:1 and even morepreferably 1:2 to 2:1 and most preferably 1:1.5 to 1.5:1. In aparticular example, the Bifidobacteria to Lactobacilli ratio is 1:1.

In particular, when Bifidobacteria animalis subsp. lactis strain 420(B420) bacteria are used in the present invention together withLactobacillus acidophilus strain NCFM bacteria, the bacteria may bepresent in any ratio capable of achieving the desired effects of theinvention described herein. Typically, the ratio of Bifidobacteriaanimalis subsp. lactis strain 420 to Lactobacillus acidophilus strainNCFM (measured in terms of colony forming units) is in the range is inthe range 1:100 to 100:1, suitably 1:50 to 50:1, preferably 1:20 to20:1, more preferably 1:10 to 10:1, still more preferably 1:5 to 5:1,yet more preferably 1:3 to 3:1 and even more preferably 1:2 to 2:1 andmost preferably 1:1.5 to 1.5:1. In a particular example, theBifidobacteria animalis subsp. lactis strain 420 to Lactobacillusacidophilus strain NCFM ratio is 1:1.

Subjects/Medical Indications

The Bifidobacteria (and, if present, the Lactobacilli) to which thepresent invention relates are administered to a mammal, including forexample livestock (including cattle, horses, pigs, chickens and sheep),and humans. In some aspects of the present invention the mammal is acompanion animal (including pets), such as a dog or a cat for instance.In some aspects of the present invention, the subject may suitably be ahuman.

The Bifidobacteria (and, if present, the Lactobacilli) to which thepresent invention relates may be suitable for treating a number ofdiseases or conditions in mammals (particularly humans). In thisspecification the term “treatment” or “treating” refers to anyadministration of the Bifidobacteria (and, if present, Lactobacilli)according to the present invention and includes: (1) preventing thespecified disease from occurring in a mammal which may be predisposed tothe disease but does not yet experience or display the pathology orsymptomatology of the disease (including prevention of one or more riskfactors associated with the disease); (2) inhibiting the disease in amammal that is experiencing or displaying the pathology orsymptomatology of the diseased (i.e., arresting further development ofthe pathology and/or symptomatology), or (3) ameliorating the disease ina mammal that is experiencing or displaying the pathology orsymptomatology of the diseased (i.e., reversing the pathology and/orsymptomatology).

The Bifidobacteria to which the present invention relates are suitablefor administration to both diabetic and obese mammals. They could alsobe suitable for diabetic and non-obese mammals, as well as to obesemammals possessing the risk factors for diabetes, but not yet in adiabetic state. This aspect is discussed in more detail below.

In particular, the use of Bifidobacteria according to the presentinvention is suitable for the treatment of mammals ingesting a high-fatdiet. This aspect is discussed in more detail below.

As described in more detail in the Examples below, the Bifidobacteriaused in the present invention have a number of biological activities. Inparticular, the Bifidobacteria used in the present invention are capableof normalising insulin sensitivity, increasing fed insulin secretion,decreasing fasted insulin secretion, improving glucose tolerance in amammal. These effects confer the potential for use in the treatment ofdiabetes and diabetes-related conditions (in particular, Type 2 diabetesand impaired glucose tolerance).

In particular, as described in more detail in the Examples below, theBifidobacteria used in combination with Lactobacillus bacteria(particularly Lactobacillus acidophilus bacteria) in accordance with thepresent invention have a number of biological activities. In particular,the Bifidobacteria used in the present invention are capable ofincreasing fed insulin secretion and improving glucose tolerance in amammal. These effects confer the potential for use in the treatment ofdiabetes and diabetes-related conditions (in particular, Type 2 diabetesand impaired glucose tolerance).

In this specification the term ‘diabetes’ includes all forms of diabeteswhich, as noted above, is characterised by disordered metabolism andabnormally high blood sugar (hyperglycaemia) resulting from insufficientlevels of the hormone insulin. The term therefore includes Type 1diabetes, Type 2 diabetes, gestational diabetes, and impaired glucosetolerance. Type 1 diabetes is characterised by loss of theinsulin-producing beta cells of the islets of Langerhans in thepancreas, leading to a deficiency of insulin. Type 2 diabetes mellitusis characterised by insulin resistance or reduced insulin sensitivity,combined with reduced insulin secretion. Gestational diabetes isformally defined as “any degree of glucose intolerance with onset orfirst recognition during pregnancy”. Impaired Glucose Tolerance (IGT) isa pre-diabetic state of dysglycemia that is associated with insulinresistance and increased risk of cardiovascular pathology. According tothe criteria of the World Health Organization and the American DiabetesAssociation, impaired glucose tolerance is defined as two-hour glucoselevels of 140 to 199 mg per dL (7.8 to 11.0 mmol) on the 75-g oralglucose tolerance test. A patient is said to be under the condition ofIGT when he/she has an intermediately raised glucose level after 2hours, but less than would qualify for type 2 diabetes mellitus. Thefasting glucose may be either normal or mildly elevated. IGT may precedetype 2 diabetes mellitus by many years. IGT is also a risk factor formortality.

In addition, the Bifidobacteria used in the present invention arecapable of inducing weight loss and lowering body fat mass (inparticular, mesenteric fat mass). These effects confer the potential foruse in the treatment of obesity and controlling weight gain and/orinducing weight loss in a mammal.

In particular, as described in more detail in the Examples below, theBifidobacteria used in combination with Lactobacillus bacteria(particularly Lactobacillus acidophilus bacteria) in accordance with thepresent invention are capable of inducing weight loss and lowering bodyfat mass (in particular, mesenteric fat mass). These effects confer thepotential for use in the treatment of obesity and controlling weightgain and/or inducing weight loss in a mammal.

In this specification, the term obesity is linked to body mass index(BMI). The body mass index (BMI) (calculated as weight in kilogramsdivided by the square of height in metres) is the most commonly acceptedmeasurement for overweight and/or obesity. A BMI exceeding 25 isconsidered overweight. Obesity is defined as a BMI of 30 or more, with aBMI of 35 or more considered as serious comorbidity obesity and a BMI of40 or more considered morbid obesity.

As noted above, the term “obesity” as used herein includes obesity,comorbidity obesity and morbid obesity. Therefore, the term “obese” asused here may be defined as a subject having a BMI of more than or equalto 30. In some embodiments, suitably an obese subject may have a BMI ofmore than or equal to 30, suitably 35, suitably 40.

While the composition of the invention is particularly suitable for usein patients who are both diabetic and obese, the composition is alsosuitable for those who are diabetic but not obese. It may also besuitable for use in obese patients possessing the risk factors fordiabetes, but not yet in a diabetic state, as it could be expected thatan obese person (but not diabetic), could limit the metabolicconsequences of his obesity, i.e. the diabetes or at leastinsulino-resistance development.

In addition, the Bifidobacteria used in the present invention may beused for treating metabolic syndrome in a mammal. Metabolic syndrome isa combination of medical disorders that increase the risk of developingcardiovascular disease and diabetes. Metabolic syndrome is also known asmetabolic syndrome X, syndrome X, insulin resistance syndrome, Reaven'ssyndrome or CHAOS (Australia).

There is currently no single accepted definition of metabolic syndrome.The World Health Organization criteria (1999) require presence ofdiabetes mellitus, impaired glucose tolerance, impaired fasting glucoseor insulin resistance, AND two of the following:

blood pressure: ≧140/90 mmHg

dyslipidaemia: triglycerides (TG): ≧1.695 mmol/L and high-densitylipoprotein cholesterol (HDL-C)≦0.9 mmol/L (male), ≦1.0 mmol/L (female)

central obesity: waist:hip ratio>0.90 (male); >0.85 (female), and/orbody mass index>30 kg/m²

microalbuminuria: urinary albumin excretion ratio≧20 mg/min or albumin:creatinine ratio≧30 mg/g.

The European Group for the Study of Insulin Resistance (1999) requiresinsulin resistance defined as the top 25% of the fasting insulin valuesamong non-diabetic individuals AND two or more of the following:

central obesity: waist circumference≧94 cm (male), ≧80 cm (female)

dyslipidaemia: TG≧2.0 mmol/L and/or HDL=C<1.0 mg/dL or treated fordyslipidaemia

hypertension: blood pressure≧140/90 mmHg or antihypertensive medicationfasting plasma glucose≧6.1 mmol/L

The US National Cholesterol Education Program (NCEP) Adult TreatmentPanel III (2001) requires at least three of the following:

central obesity: waist circumference≧102 cm or 40 inches (male), ≧88 cmor 36 inches (female)

dyslipidaemia: TG≧1.695 mmol/L (150 mg/dl)

dyslipidaemia: HDL-C<40 mg/dL (male), <50 mg/dL (female)

blood pressure≧130/85 mmHg

fasting plasma glucose≧6.1 mmol/L (110 mg/dl)

In further embodiments, the Bifidobacteria (and, if present, theLactobacilli) used in the present invention may be used to lower tissueinflammation (particularly, although not exclusively, liver tissueinflammation, muscle tissue inflammation and/or adipose tissueinflammation) in a mammal.

In one embodiment, the Bifidobacteria (and, if present, theLactobacilli) used in the present invention may be used to lower livertissue inflammation. This confers the potential for the application ofthe bacteria in the treatment of hepatitis, which is characterised bythe destruction of a number of liver cells and the presence ofinflammatory cells in the liver tissue.

Hepatitis can be divided into two subgroups according to its duration:acute hepatitis (lasting less than six months) and chronic hepatitis(lasting longer than six months). Hepatitis may be also classifiedaccording to its cause: for example, hepatitis may comprise Infectiousviral hepatitis (such as hepatitis A, hepatitis B, hepatitis C,hepatitis D and hepatitis E), hepatitis caused by other viral diseases(such as mononucleosis and cytomegalovirus), hepatitis caused by severebacterial infections or amoebic infections. hepatitis caused bymedicines, hepatitis caused by toxins such as alcohol, autoimmunehepatitis (in which a number of liver cells are destroyed by thepatient's own immune system) and hepatitis caused by congenitalmetabolic disorders, such as Wilson's disease (disorder of the body'scopper etabolism) and haemochromatosis (disorder of the body's ironmetabolism).

In one embodiment, the Bifidobacteria (and, if present, theLactobacilli) used in the present invention may be used to lower muscletissue inflammation. This confers the potential for the application ofthe bacteria in the treatment of myositis, in which the muscle fibersand skin are inflamed and damaged, resulting in muscle weakness.

There are several types of myositis that affect different parts of thebody. Particular forms of myositis treatable according to the presentinvention include: polymyositis (PM) (in which muscles in many parts ofthe body, and especially those parts closest to the trunk, areinflamed); dermatomyositis (DM) (which affects both the muscle fibersand skin by damaging capillaries that supply blood to the muscle andskin), inclusion body myositis (IBM) which is characterized by gradualweakening of muscles throughout the body, including the wrists orfingers, development of dysphagia, and atrophy of forearms and/or thighmuscles; and juvenile myositis (JM), which involves muscle weakness,skin rash, and dysphagia in children.

The present inventors have surprisingly found that the Bifidobacteria(and, if present, the Lactobacilli) to which the present inventionrelates are capable of lowering adipose tissue inflammation in mammals.There is epidemiological evidence in the literature showing astatistical relationship between inflammation, obesity and insulinresistance in humans (Cani et al., Diabetes, 2007, 56, 1761-1772, andreferences cited therein). This finding therefore confers the potentialfor the Bifidobacteria (and, if present, the Lactobacilli) to be usefulin the treatment of obesity, diabetes and related conditions, metabolicdiseases and cardiovascular consequences in mammals.

According to Berg and Scherer, Circulation Research, 2005, 96, 939,recent evidence highlights the role of adipose tissue in the developmentof a systemic inflammatory state that contributes to obesity-associatedvasculopathy and cardiovascular risk. Circulating mediators ofinflammation participate in the mechanisms of vascular insult andatheromatous change, and many of these inflammatory proteins aresecreted directly from adipocytes and adipose tissue-derivedmacrophages. Several factors linking obesity with an increasedcardiovascular risk have been identified. The adipocyte-specificsecretory protein adiponectin is a particularly promising candidate inthis context. Its levels are decreased in obesity.

The targeted suppression of various proinflammatory cascades inadipocytes specifically represents a new therapeutic opportunity for thecardiovascular disease area. Suppression of adipose tissue inflammationwould therefore be expected to provide a therapeutic benefit in thetreatment of cardiovascular diseases.

Examples of cardiovascular diseases treatable by use of theBifidobacteria (and, if present, the Lactobacilli) according to thepresent invention include aneurysm, angina, atherosclerosis,cerebrovascular accident (stroke), cerebrovascular disease, congestiveheart failure (CHF), coronary artery disease, myocardial infarction(heart attack) and peripheral vascular disease.

An aneurysm is a localized, blood-filled dilation (balloon-like bulge)of a blood vessel caused by disease or weakening of the vessel wall.Aneurysms most commonly occur in arteries at the base of the brain (thecircle of Willis) and in the aorta (the main artery coming out of theheart, a so-called aortic aneurysm). As the size of an aneurysmincreases, there is an increased risk of rupture, which can result insevere hemorrhage or other complications including sudden death.

Angina pectoris, commonly known as angina, is severe chest pain due toischemia (a lack of blood and hence oxygen supply) of the heart muscle,generally due to obstruction or spasm of the coronary arteries (theheart's blood vessels). Coronary artery disease, the main cause ofangina, is due to atherosclerosis of the cardiac arteries.

Atherosclerosis is the condition in which an artery wall thickens as theresult of a build up of fatty materials such as cholesterol. It is asyndrome affecting arterial blood vessels. It is a chronic inflammatoryresponse in the walls of arteries, in large part due to the accumulationof macrophage white blood cells and promoted by low density (especiallysmall particle) lipoproteins (plasma proteins that carry cholesterol andtriglycerides) without adequate removal of fats and cholesterol from themacrophages by functional high density lipoproteins (HDL). It iscommonly referred to as a hardening or furring of the arteries. It iscaused by the formation of multiple plaques within the arteries.

A stroke is the rapidly developing loss of brain function(s) due todisturbance in the blood supply to the brain. This can be due toischemia (lack of blood supply) caused by thrombosis or embolism or dueto a hemorrhage. As a result, the affected area of the brain is unableto function, leading to inability to move one or more limbs on one sideof the body, inability to understand or formulate speech, or see oneside of the visual field and ultimately to death.

Cerebrovascular disease is a group of brain dysfunctions related todisease of blood vessels supplying the brain. Hypertension is the mostimportant cause that damages the blood vessel lining endotheliumexposing the underlying collagen where platelets aggregate to initiate arepairing process which is not always complete and perfect. Sustainedhypertension permanently changes the architecture of the blood vesselsmaking them narrow, stiff, deformed and uneven which are more vulnerableto fluctuations of blood pressure. A fall in blood pressure during sleepcan lead to marked reduction in blood flow in the narrowed blood vesselscausing ischemic stroke in the morning whereas a sudden rise in bloodpressure can cause tearing of the blood vessels causing intracranialhemorrhage during excitation at daytime. Primarily people who areelderly, diabetic, smoker, or have ischemic heart disease, havecerebrovascular disease. All diseases related to artery dysfunction canbe classified under a disease as known as macrovascular disease. This isa simplistic study by which arteries are blocked by fatty deposits or bya blood clot. The results of cerebrovascular disease can include astroke, or even sometimes a hemorrhagic stroke. Ischemia or other bloodvessel dysfunctions can affect one during a cerebrovascular accident.

Heart failure is a global term for the physiological state in whichcardiac output is insufficient for the body's needs. This may occur whenthe cardiac output is low (often termed “congestive heart failure”).Common causes of heart failure include myocardial infarction and otherforms of ischemic heart disease, hypertension, valvular heart diseaseand cardiomyopathy.

Coronary disease (or coronary heart disease) refers to the failure ofcoronary circulation to supply adequate circulation to cardiac muscleand surrounding tissue. It is most commonly equated with atheroscleroticcoronary artery disease, but coronary disease can be due to othercauses, such as coronary vasospasm. It is possible for the stenosis tobe caused by the spasm.

Myocardial infarction, commonly known as a heart attack, occurs when theblood supply to part of the heart is interrupted causing some heartcells to die. This is most commonly due to occlusion (blockage) of acoronary artery following the rupture of a vulnerable atheroscleroticplaque, which is an unstable collection of lipids (like cholesterol) andwhite blood cells (especially macrophages) in the wall of an artery. Theresulting ischemia (restriction in blood supply) and oxygen shortage, ifleft untreated for a sufficient period of time, can cause damage and/ordeath (infarction) of heart muscle tissue (myocardium).

Peripheral vascular disease (PVD), also known as peripheral arterydisease (PAD) or peripheral artery occlusive disease (PAOD), includesall diseases caused by the obstruction of large arteries in the arms andlegs. PVD can result from atherosclerosis, inflammatory processesleading to stenosis, an embolism or thrombus formation. It causes eitheracute or chronic ischemia (lack of blood supply), typically of the legs.

It is envisaged within the scope of the present invention that theembodiments of the invention can be combined such that combinations ofany of the features described herein are included within the scope ofthe present invention. In particular, it is envisaged within the scopeof the present invention that any of the therapeutic effects of thebacteria may be exhibited concomitantly.

Diet

As noted above, diabetic and/or obese mammals treated with bacteriaaccording to the present invention may ingest a high-fat diet whilemitigating the metabolic consequences of their condition(s). In thisspecification the term ‘high-fat diet’ means a diet generally containingat least 20%, preferably at least 25%, such as at least 30%, for exampleat least 35%, such as at least 40%, for example at least 45%, such as atleast 50%, for example at least 55%, such as at least 60%, for exampleat least 65%, such as at least 70%, for example at least 75%, such as atleast 80%, for example at least 85%, such as at least 90% of caloriesfrom fat.

In some embodiments, mammals treated with bacteria according to thepresent invention may ingest a low-carbohydrate diet during the courseof the treatment. In this specification the term ‘low-carbohydrate diet’means a diet generally containing no greater than 50%, such as nogreater than 45%, for example no greater than 40%, such as no greaterthan 35%, for example no greater than 30%, such as no greater than 25%,for example no greater than 20%, such as no greater than 15%, forexample no greater than 10%, such as no greater than 5%, for example nogreater than 2%, such as no greater than 1%, for example no greater than0.5%, such as no greater than 0.2% of calories from carbohydrate.

Compositions

While is it possible to administer Bifidobacteria (and, if present,Lactobacilli) alone according to the present invention (i.e. without anysupport, diluent or excipient), the Bifidobacteria (and, if present,Lactobacilli bacteria) are typically and preferably administered on orin a support as part of a product, in particular as a component of afood product, a dietary supplement or a pharmaceutical formulation.These products typically contain additional components well known tothose skilled in the art.

Any product which can benefit from the composition may be used in thepresent invention. These include but are not limited to foods,particularly fruit conserves and dairy foods and dairy food-derivedproducts, and pharmaceutical products. The Bifidobacteria (and, ifpresent, Lactobacilli) may be referred to herein as “the composition ofthe present invention” or “the composition”.

Food

In one embodiment, the Bifidobacteria (and, if present, Lactobacillibacteria) are employed according to the invention in a food product suchas a food supplement, a drink or a powder based on milk. Here, the term“food” is used in a broad sense and covers food for humans as well asfood for animals (i.e. a feed). In a preferred aspect, the food is forhuman consumption.

The food may be in the form of a solution or as a solid—depending on theuse and/or the mode of application and/or the mode of administration.

When used as, or in the preparation of, a food, such as functional food,the composition of the present invention may be used in conjunction withone or more of: a nutritionally acceptable carrier, a nutritionallyacceptable diluent, a nutritionally acceptable excipient, anutritionally acceptable adjuvant, a nutritionally active ingredient.

By way of example, the composition of the present invention can be usedas an ingredient to soft drinks, a fruit juice or a beverage comprisingwhey protein, health teas, cocoa drinks, milk drinks and lactic acidbacteria drinks, yoghurt and drinking yoghurt, cheese, ice cream, waterices and desserts, confectionery, biscuits cakes and cake mixes, snackfoods, balanced foods and drinks, fruit fillings, care glaze, chocolatebakery filling, cheese cake flavoured filling, fruit flavoured cakefilling, cake and doughnut icing, instant bakery filling creams,fillings for cookies, ready-to-use bakery filling, reduced caloriefilling, adult nutritional beverage, acidified soy/juice beverage,aseptic/retorted chocolate drink, bar mixes, beverage powders, calciumfortified soy/plain and chocolate milk, calcium fortified coffeebeverage.

The composition can further be used as an ingredient in food productssuch as American cheese sauce, anti-caking agent for grated & shreddedcheese, chip dip, cream cheese, dry blended whip topping fat free sourcream, freeze/thaw dairy whipping cream, freeze/thaw stable whippedtipping, low fat and light natural cheddar cheese, low fat Swiss styleyoghurt, aerated frozen desserts, hard pack ice cream, label friendly,improved economics & indulgence of hard pack ice cream, low fat icecream: soft serve, barbecue sauce, cheese dip sauce, cottage cheesedressing, dry mix Alfredo sauce, mix cheese sauce, dry mix tomato sauceand others.

The term “dairy product” as used herein is meant to include a mediumcomprising milk of animal and/or vegetable origin. As milk of animalorigin there can be mentioned cow's, sheep's, goat's or buffalo's milk.As milk of vegetable origin there can be mentioned any fermentablesubstance of vegetable origin which can be used according to theinvention, in particular originating from soybeans, rice or cereals.

Still more preferably the food product employed according to theinvention is a fermented milk or humanized milk.

For certain aspects, preferably the present invention may be used inconnection with yoghurt production, such as fermented yoghurt drink,yoghurt, drinking yoghurt, cheese, fermented cream, milk based dessertsand others.

Suitably, the composition can be further used as an ingredient in one ormore of cheese applications, meat applications, or applicationscomprising protective cultures.

The present invention also provides a method of preparing a food or afood ingredient, the method comprising admixing the compositionaccording to the present invention with another food ingredient.

Advantageously, the present invention relates to products that have beencontacted with the composition of the present invention (and optionallywith other components/ingredients), wherein the composition is used inan amount to be capable of improving the nutrition and/or healthbenefits of the product.

As used herein the term “contacted” refers to the indirect or directapplication of the composition of the present invention to the product.Examples of the application methods which may be used, include, but arenot limited to, treating the product in a material comprising thecomposition, direct application by mixing the composition with theproduct, spraying the composition onto the product surface or dippingthe product into a preparation of the composition.

Where the product of the invention is a foodstuff, the composition ofthe present invention is preferably admixed with the product.Alternatively, the composition may be included in the emulsion or rawingredients of a foodstuff. In a further alternative, the compositionmay be applied as a seasoning, glaze, colorant mixture, and the like.

For some applications, it is important that the composition is madeavailable on or to the surface of a product to be affected/treated. Thisallows the composition to impart one or more of the following favourablecharacteristics: nutrition and/or health benefits.

The compositions of the present invention may be applied to intersperse,coat and/or impregnate a product with a controlled amount of amicroorganism.

Preferably, the composition is used to ferment milk or sucrose fortifiedmilk or lactic media with sucrose and/or maltose where the resultingmedia containing all components of the composition—i.e. saidmicroorganism according to the present invention—can be added as aningredient to yoghurt milk in suitable concentrations—such as forexample in concentrations in the final product which offer a daily doseof 10⁶-10¹⁰ cfu. The microorganism according to the present inventionmay be used before or after fermentation of the yoghurt.

For some aspects the microorganisms according to the present inventionare used as, or in the preparation of, animal feeds, such as livestockfeeds, in particular poultry (such as chicken) feed, or pet food.

Advantageously, where the product is a food product, the Bifidobacteria(and, if present, Lactobacilli) should remain effective through thenormal “sell-by” or “expiration” date during which the food product isoffered for sale by the retailer. Preferably, the effective time shouldextend past such dates until the end of the normal freshness period whenfood spoilage becomes apparent. The desired lengths of time and normalshelf life will vary from foodstuff to foodstuff and those of ordinaryskill in the art will recognise that shelf-life times will vary upon thetype of foodstuff, the size of the foodstuff, storage temperatures,processing conditions, packaging material and packaging equipment.

Food Ingredient

The composition of the present invention may be used as a foodingredient and/or feed ingredient.

As used herein the term “food ingredient” or “feed ingredient” includesa formulation which is or can be added to functional foods or foodstuffsas a nutritional supplement.

The food ingredient may be in the form of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

Food Supplements

The composition of the present invention may be—or may be added to—foodsupplements (also referred to herein as dietary supplements).

Functional Foods

The composition of the present invention may be—or may be addedto—functional foods.

As used herein, the term “functional food” means food which is capableof providing not only a nutritional effect, but is also capable ofdelivering a further beneficial effect to consumer.

Accordingly, functional foods are ordinary foods that have components oringredients (such as those described herein) incorporated into them thatimpart to the food a specific functional—e.g. medical or physiologicalbenefit—other than a purely nutritional effect.

Although there is no legal definition of a functional food, most of theparties with an interest in this area agree that they are foods marketedas having specific health effects beyond basic nutritional effects.

Some functional foods are nutraceuticals. Here, the term “nutraceutical”means a food which is capable of providing not only a nutritional effectand/or a taste satisfaction, but is also capable of delivering atherapeutic (or other beneficial) effect to the consumer. Nutraceuticalscross the traditional dividing lines between foods and medicine.

Medicament

The term “medicament” as used herein encompasses medicaments for bothhuman and animal usage in human and veterinary medicine. In addition,the term “medicament” as used herein means any substance which providesa therapeutic and/or beneficial effect. The term “medicament” as usedherein is not necessarily limited to substances which need MarketingApproval, but may include substances which can be used in cosmetics,nutraceuticals, food (including feeds and beverages for example),probiotic cultures, and natural remedies. In addition, the term“medicament” as used herein encompasses a product designed forincorporation in animal feed, for example livestock feed and/or petfood.

Pharmaceutical

The composition of the present invention may be used as—or in thepreparation of—a pharmaceutical. Here, the term “pharmaceutical” is usedin a broad sense—and covers pharmaceuticals for humans as well aspharmaceuticals for animals (i.e. veterinary applications). In apreferred aspect, the pharmaceutical is for human use and/or for animalhusbandry.

The pharmaceutical can be for therapeutic purposes—which may be curativeor palliative or preventative in nature. The pharmaceutical may even befor diagnostic purposes.

A pharmaceutically acceptable support may be for example a support inthe form of compressed tablets, tablets, capsules, ointments,suppositories or drinkable solutions. Other suitable forms are providedbelow.

When used as—or in the preparation of—a pharmaceutical, the compositionof the present invention may be used in conjunction with one or more of:a pharmaceutically acceptable carrier, a pharmaceutically acceptablediluent, a pharmaceutically acceptable excipient, a pharmaceuticallyacceptable adjuvant, a pharmaceutically active ingredient.

The pharmaceutical may be in the form of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

The Bifidobacteria (and, if present, Lactobacilli) of the presentinvention may be used as pharmaceutical ingredients. Here, thecomposition may be the sole active component or it may be at least oneof a number (i.e. 2 or more) of active components.

The pharmaceutical ingredient may be in the form of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

The Bifidobacteria (and, if present, Lactobacilli) may be used accordingto the present invention in any suitable form—whether when alone or whenpresent in a combination with other components or ingredients. Thelactic acid bacteria used in the present invention may be referred toherein as “the composition”. Likewise, combinations comprising thecomposition of the present invention and other components and/oringredients (i.e. ingredients—such as food ingredients, functional foodingredients or pharmaceutical ingredients) may be used in any suitableform.

The Bifidobacteria (and, if present, Lactobacilli) may be used accordingto the present invention in the form of solid or liquid preparations oralternatives thereof. Examples of solid preparations include, but arenot limited to tablets, capsules, dusts, granules and powders which maybe wettable, spray-dried or freeze-dried. Examples of liquidpreparations include, but are not limited to, aqueous, organic oraqueous-organic solutions, suspensions and emulsions.

Suitable examples of forms include one or more of: tablets, pills,capsules, ovules, solutions or suspensions, which may contain flavouringor colouring agents, for immediate-, delayed-, modified-, sustained-,pulsed- or controlled-release applications.

By way of example, if the composition of the present invention is usedin a tablet form—such for use as a functional ingredient—the tablets mayalso contain one or more of: excipients such as microcrystallinecellulose, lactose, sodium citrate, calcium carbonate, dibasic calciumphosphate and glycine; disintegrants such as starch (preferably corn,potato or tapioca starch), sodium starch glycollate, croscarmellosesodium and certain complex silicates; granulation binders such aspolyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),hydroxypropylcellulose (HPC), sucrose, gelatin and acacia; lubricatingagents such as magnesium stearate, stearic acid, glyceryl behenate andtalc may be included.

Examples of nutritionally acceptable carriers for use in preparing theforms include, for example, water, salt solutions, alcohol, silicone,waxes, petroleum jelly, vegetable oils, polyethylene glycols, propyleneglycol, liposomes, sugars, gelatin, lactose, amylose, magnesiumstearate, talc, surfactants, silicic acid, viscous paraffin, perfumeoil, fatty acid monoglycerides and diglycerides, petroethral fatty acidesters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

Preferred excipients for the forms include lactose, starch, a cellulose,milk sugar or high molecular weight polyethylene glycols.

For aqueous suspensions and/or elixirs, the composition of the presentinvention may be combined with various sweetening or flavouring agents,colouring matter or dyes, with emulsifying and/or suspending agents andwith diluents such as water, propylene glycol and glycerin, andcombinations thereof.

The forms may also include gelatin capsules; fibre capsules, fibretablets etc.; or even fibre beverages.

Further examples of form include creams. For some aspects themicroorganism used in the present invention may be used inpharmaceutical and/or cosmetic creams such as sun creams and/orafter-sun creams for example.

In one aspect, the composition according to the present invention may beadministered in an aerosol, for example by way of a nasal spray, forinstance for administration to the respiratory tract.

Combinations

The composition of the present invention may additionally contain one ormore prebiotics. Prebiotics are a category of functional food, definedas non-digestible food ingredients that beneficially affect the host byselectively stimulating the growth and/or activity of one or a limitednumber of bacteria (particularly, although not exclusively, probiotics,Bifidobacteria and/or lactic acid bacteria) in the colon, and thusimprove host health. Typically, prebiotics are carbohydrates (such asoligosaccharides), but the definition does not precludenon-carbohydrates. The most prevalent forms of prebiotics arenutritionally classed as soluble fibre. To some extent, many forms ofdietary fibre exhibit some level of prebiotic effect.

In one embodiment, a prebiotic is a selectively fermented ingredientthat allows specific changes, both in the composition and/or activity inthe gastrointestinal microflora that confers benefits upon hostwell-being and health.

Suitably, the prebiotic may be used according to the present inventionin an amount of 0.01 to 100 g/day, preferably 0.1 to 50 g/day, morepreferably 0.5 to 20 g/day. In one embodiment, the prebiotic may be usedaccording to the present invention in an amount of 1 to 100 g/day,preferably 2 to 9 g/day, more preferably 3 to 8 g/day. In anotherembodiment, the prebiotic may be used according to the present inventionin an amount of 5 to 50 g/day, preferably 10 to 25 g/day.

Examples of dietary sources of prebiotics include soybeans, inulinsources (such as Jerusalem artichoke, jicama, and chicory root), rawoats, unrefined wheat, unrefined barley and yacon.

Examples of suitable prebiotics include alginate, xanthan, pectin,locust bean gum (LBG), inulin, guar gum, galacto-oligosaccharide (GOS),fructo-oligosaccharide (FOS), polydextrose (i.e. Litesse®), lactitol,lactosucrose, soybean oligosaccharides, isomaltulose (Palatinose™),isomalto-oligosaccharides, gluco-oligosaccharides,xylo-oligosaccharides, manno-oligosaccharides, beta-glucans, cellobiose,raffinose, gentiobiose, melibiose, xylobiose, cyclodextrins, isomaltose,trehalose, stachyose, panose, pullulan, verbascose, galactomannans, andall forms of resistant starches. A particularly preferred example of aprebiotic is polydextrose.

In some embodiments, a combination of Bifidobacterium (and, if present,Lactobacillus) bacteria and prebiotics according to the presentinvention exhibits a synergistic effect in certain applications (i.e. aneffect which is greater than the additive effect of the bacteria whenused separately). Without wishing to be bound by theory, it is believedthat such a combination is capable of selectively stimulating the growthand/or activity of the Bifidobacteria (and, if present, Lactobacilli)bacteria in the colon, and thus improve host health.

In one embodiment, the Bifidobacteria (and, if present, Lactobacilli)may be used according to the present invention in combination with oneor more antidiabetic drugs. Examples of oral antidiabetic drugs whichmay be used in such a combination include biguanides (such asmetformin), sulfonylureas (such as carbutamide, chlorpropamide,glibenclamide (Glyburide™), gliclazide, glimepiride, glipizide,gliquidone, tolazamide or tolbutamide), alpha-glucosidase inhibitors(such as acarbose, miglitol or voglibose), thiazolidinediones (TZD)(such as pioglitazone, rivoglitazone or rosiglitazone), meglitinides(such as nateglinide, repaglinide or mitiglinide), dipeptidylpeptidase-4 (DPP-4) inhibitors (such as alogliptin, saxagliptin,sitagliptin or vildagliptin), glucagon-like peptide-1 analogs (such asexenatide, liraglutide, or albiglutide), amylin analogs (such aspramlintide), fast acting insulin analogs (such as insulin lispro,insulin aspart and insulin glulisine), long acting insulin analogs (suchas insulin glargine, insulin detemir), dual PPAR agonists (such asaleglitazar) and SGLT2 inhibitors (such as dapagliflozin, remogliflozinand sergliflozin). A particularly preferred example is metformin.

The dosage, mode of administration and formulation of the aboveantidiabetic drugs for use in the combination of the present inventionwill be readily apparent to a skilled person. Suitably, the antidiabeticdrug may be used according to the present invention in an amount of 1 μgto 10 g/day, preferably 10 μg to 5 g/day, more preferably 0.1 mg to 2g/day. In one embodiment, the antidiabetic drug may be used according tothe present invention in an amount of 1 mg to 1 g/day, preferably 5 to500 mg/day.

In one embodiment, the Bifidobacteria (and, if present, Lactobacilli)may be used according to the present invention in combination with botha prebiotic (as described and exemplified above) and an antidiabeticdrug (as described and exemplified above).

Preferably, the Bifidobacterium used in the combination (with aprebiotic, an antidiabetic drug, or both) is of the speciesBifidobacterium animalis. More preferably, the Bifidobacterium used inthe combination is of the species Bifidobacterium animalis subsp.lactis. In a particularly preferred embodiment, the bacteria used in thecombination are Bifidobacterium animalis subsp. lactis strain 420(B420).

Suitably, the prebiotic used in the combination is polydextrose.

Suitably, the antidiabetic used in the combination is metformin.

In a particularly preferred embodiment, the bacteria used in thecombination are Bifidobacterium animalis subsp. lactis strain 420(B420), the prebiotic is polydextrose and the antidiabetic is metformin.

In another embodiment, a prebiotic may be used according to the presentinvention in combination with an antidiabetic drug, but in the absenceof Bifidobacteria, Lactobacilli or other bacteria used in the otherembodiments of this invention.

Therefore, in a further aspect, the invention comprises a combination ofa prebiotic or a mixture thereof and an antidiabetic drug or a mixturethereof.

In a yet further aspect, the invention comprises a food product or foodproduct intermediate including a prebiotic or a mixture thereof and anantidiabetic drug or a mixture thereof.

In a yet further aspect, the invention comprises a pharmaceuticalcomposition comprising a prebiotic or a mixture thereof and anantidiabetic drug or a mixture thereof, together with a pharmaceuticallyacceptable carrier or diluent.

In one aspect, the invention comprises use of a combination of aprebiotic or a mixture thereof and an antidiabetic drug or a mixturethereof in the manufacture of a food product, dietary supplement ormedicament for treating diabetes (preferably but not exclusively Type 2diabetes) in a mammal.

In another aspect, the invention comprises use of a combination of aprebiotic or a mixture thereof and an antidiabetic drug or a mixturethereof in the manufacture of a food product, dietary supplement ormedicament for treating impaired glucose tolerance in a mammal.

In a further aspect, the invention comprises use of a combination of aprebiotic or a mixture thereof and an antidiabetic drug or a mixturethereof in the manufacture of a food product, dietary supplement ormedicament for normalising insulin sensitivity in a mammal.

In a yet further aspect, the invention comprises use of a combination ofa prebiotic or a mixture thereof and an antidiabetic drug or a mixturethereof in the manufacture of a food product, dietary supplement ormedicament for increasing fed insulin secretion in a mammal.

In a still further aspect, the invention comprises use of a combinationof a prebiotic or a mixture thereof and an antidiabetic drug or amixture thereof in the manufacture of a food product, dietary supplementor medicament for decreasing fasted insulin secretion in a mammal.

In an additional aspect, the invention comprises use of a combination ofa prebiotic or a mixture thereof and an antidiabetic drug or a mixturethereof in the manufacture of a food product, dietary supplement ormedicament for improving glucose tolerance in a mammal.

In a yet further aspect, the invention comprises use of a combination ofa prebiotic or a mixture thereof and an antidiabetic drug or a mixturethereof in the manufacture of a food product, dietary supplement ormedicament for treating metabolic syndrome in a mammal.

In this embodiment, examples of suitable prebiotics which may be used insuch a combination include alginate, xanthan, pectin, locust bean gum(LBG), inulin, guar gum, galacto-oligosaccharide (GOS),fructo-oligosaccharide (FOS), polydextrose (i.e. Litesse®), lactitol,lactosucrose, soybean oligosaccharides, isomaltulose (Palatinose™),isomalto-oligosaccharides, gluco-oligosaccharides,xylo-oligosaccharides, manno-oligosaccharides, beta-glucans, cellobiose,raffinose, gentiobiose, melibiose, xylobiose, cyclodextrins, isomaltose,trehalose, stachyose, panose, pullulan, verbascose, galactomannans, andall forms of resistant starches. A particularly preferred example of aprebiotic is polydextrose.

In this embodiment, examples of oral antidiabetic drugs which may beused in such a combination include biguanides (such as metformin),sulfonylureas (such as carbutamide, chlorpropamide, glibenclamide(Glyburide™), gliclazide, glimepiride, glipizide, gliquidone, tolazamideor tolbutamide), alpha-glucosidase inhibitors (such as acarbose,miglitol or voglibose), thiazolidinediones (TZD) (such as pioglitazone,rivoglitazone or rosiglitazone), meglitinides (such as nateglinide,repaglinide or mitiglinide), dipeptidyl peptidase-4 (DPP-4) inhibitors(such as alogliptin, saxagliptin, sitagliptin or vildagliptin),glucagon-like peptide-1 analogs (such as exenatide, liraglutide, oralbiglutide), amylin analogs (such as pramlintide), fast acting insulinanalogs (such as insulin lispro, insulin aspart and insulin glulisine),long acting insulin analogs (such as insulin glargine, insulin detemir),dual PPAR agonists (such as aleglitazar) and SGLT2 inhibitors (such asdapagliflozin, remogliflozin and sergliflozin). A particularly preferredexample is metformin. The dosage, mode of administration and formulationof the above antidiabetic drugs for use in the combination of thisembodiment will be readily apparent to a skilled person.

In this embodiment, the prebiotic used in the combination is preferablypolydextrose and the antidiabetic used in the combination is metformin

In this combination, suitably, the prebiotic may be used in an amount of0.01 to 100 g/day, preferably 0.1 to 50 g/day, more preferably 0.5 to 20g/day. In one embodiment, the prebiotic may be used in an amount of 1 to100 g/day, preferably 2 to 9 g/day, more preferably 3 to 8 g/day. Inanother embodiment, the prebiotic may be used in an amount of 5 to 50g/day, preferably 10 to 25 g/day.

In this combination, suitably, the antidiabetic drug may be used in anamount of 1 to 10 g/day, preferably 10 μg to 5 g/day, more preferably0.1 mg to 2 g/day. In one embodiment, the antidiabetic drug may be usedaccording to the present invention in an amount of 1 mg to 1 g/day,preferably 5 to 500 mg/day.

Example 1

Materials and Methods

Animal Model and Probiotic Treatment

A cohort of fifty C57Bl/6 10-wk-old male mice were fed a Normal Chow(NC) (A03, SAFE, Augy, France), or a high-fat diet (HFD) (comprising 72%fat (corn oil and lard), 28% protein and <1% carbohydrates) (SAFE, Augy,France) for 4 weeks. This diet has the peculiar advantage to inducediabetes before the onset of obesity (see for example Cani et al. 2008“Role of gut microflora in the development of obesity and insulinresistance following high-fat diet feeding”. Pathol Biol (Paris); Caniet al, Diabetes 2008, 57, 1470-81; Knauf et al. Endocrinology 2008, 149,4768-77; Cani et al., Diabetologia 2007, 50, 2374-83; Cani et al;Diabetes 2007, 56, 1761-1772 and Turini et al. Swiss Med Wkly 2007, 137,700-4).

The mice underwent an intraperitoneal glucose tolerance test. The areaunder curve was calculated and the mice dispatched homogeneouslyaccording to the different experimental groups or ten mice per group (10mice per group). The mice were fed four more weeks with a normal chow(n=10) or a HFD (n=40). The HFD mice were treated daily for 4 weeks asfollows with, 1. Vehicle treated, 2. Bifidobacterium animalis subsp.lactis strain 420 (B420) (10⁹/bacteria per mouse), 3. Lactobacillusacidophilus NCFM (NCFM) (10⁹/bacteria per mouse), 4. NCFM+B420(5×10^(8 B)420+5×10⁸ NCFM per mouse). An intraperitoneal test was thenperformed as described below. The mice were housed in a controlledenvironment (inverted 12-h daylight cycle, light off at 10:00 a.m.).

Weight Assessment

Mice were weighed weekly in the beginning of the study dietadministration (4 weeks prior to probiotic administration) until 6 weeksinto the probiotic treatment (until sacrifice).

Body Composition

Mouse body composition was measured monthly using ECO-MRI. Total bodyfat mass, total lean body mass, total water mass, free water content,subcutaneous adipose tissue weight, mesenteric adipose tissue weight andliver weight were measured.

Glucose Tolerance

Glucose tolerance was tested after 4-week administration the study diets(before probiotic supplementation) to ensure the glucose-intolerant anddiabetic status of the HFD mice, and after 4 weeks of probioticadministration. Briefly, six-hour-fasted mice were injected with 20%glucose (1 g/kg) into the peritoneal cavity. Glycemia was determinedwith a glucose meter (ACCU-check Active, Roche, Meylan, France) at 30minutes before the glucose challenge, at the time of the glucosechallenge, and 30, 60 and 90 minutes after the glucose challenge, from3.5 μL of tail-vein tip collected blood.

Plasma Insulin

Insulin concentration was measured from plasma in fasted state as wellas in fed state.

Insulin Sensitivity

At completion of the probiotic treatment mice underwent an intrafemoralsurgery where a catheter was indwelled for further intravenousinfusions. This intravenous intrafemoral catheter was implanted 4 daysbefore the beginning of the experimental day (infusions). The day of theassay the mice were fasted for 5 hours. A hyperinsulinemic euglycemicclamp was performed for 3 hours in the presence of tritiated labeledglucose to determine the glucose turnover rate. Cold glucose wascoinfused to maintain euglycemia.

Inflammatory Markers (Real-Time Quantitative PCR)

The inflammation status of adipose, liver and muscle tissue was measuredby measuring the concentration of inflammatory markers TNFα, IL-1β,PAI-1, IL6 mRNAs by quantitative RT-PCR analysis. Total mRNAs from thegrafted fat pads and the recipient subcutaneous adipose, liver andmuscle tissue were extracted using TriPure reagent (Roche, Basel,Switzerland). PCRs were performed using an AbiPrism 7900 SequenceDetection System instrument and software (Applied Biosystems, FosterCity, Calif., USA, as described in Cani et al. Diabetes 2007, 56,1761-1772. The concentration of each mRNA was normalized for RNA loadingfor each sample using RPL19 rRNA as an internal standard.

Results

Glucose Tolerance

As shown in FIG. 1, all groups of mice fed a high fat diet for 4 weekswere glucose intolerant and diabetic. Following B420 treatment, micewere characterized by an improved glucose tolerance (FIG. 2).Significant decrease is achieved with B420 only; an trend towards healthbenefit was obtained for NCFM alone or with a combination of NCFM andB420. Therefore it was concluded that the B420 treatment began toimprove glucose tolerance. A longer period of treatment could have had agreater impact on the glycemic profiles.

Mouse Body Composition

The data show that four weeks of probiotic treatment with B420 and acombination of B420 and NCFM reduced the impact of HFD on body fat massincrease (FIG. 3). No effect of the treatment on lean body mass, freewater mass, and total water mass were observed (FIGS. 4, 5 and 6).

In particular, FIG. 3 illustrates the effect of B420 treatment andcombination of B420+NCFM in reducing the impact of high fat diet on theincreased body fat mass (wherein the left column signifies the resultbefore treatment and the right column that after treatment). In FIG.3, * indicates a result significantly different from non treated mice ofthe same group. A small increase of weight was observed with B420 orwith the combination of B420 and NCFM. However, this represents asignificant improvement on the untreated HFD mice, as the HFD resultsshow that ingestion of the HFD should increase significantly the bodyfat mass of the mice.

As the treatment showed no effect on lean body mass, free water mass,and total water mass, it can be concluded from the above that thedifference in weight achieved is only related to adipose tissues.

Individual Tissue Weight

The data show that the mesenteric adipose tissue weight was reduced byB420 and B420+NCFM. No differences in subcutaneous adipose tissue or theliver weight were noted (FIGS. 7, 8 and 9).

In particular, FIG. 8 illustrates the effect on mesenteric adiposetissue weight in high-fat diet fed mice treated with probiotics.Significant were achieved for B420 alone or in combination with NCFM (aP value of <0.05 was achieved when compared with the untreated HFDgroup). The B420 and B420+NCFM treated mice were characterized with lessmesenteric fat mass.

In addition, FIG. 9 illustrates the effect on liver weight in high fatdiet fed mice treated with probiotics. No significant difference wasnoted, once again showing that the weight difference is not linked withthe liver weight.

Weight Gain

FIG. 10 illustrates the body weight gain before and after probioticadministration of high fat diet fed mice. Body weight gain of the B420and the B420+NCFM treated mice was lower than that of the mice fed HFDwithout probiotics or mice treated with NCFM alone. Statisticallysignificant results were achieved for B420 alone or in combination withNCFM; a downward trend (in comparison with untreated HFD-fed mice) wasobserved for NCFM alone.

Plasma Insulin Concentrations

Plasma insulin concentration was assessed in the fasted and the fedstate. The data show that, in fasting state, the B420 treated group wascharacterized by a normalization of fasting hyperinsulinemia (FIG. 11).In the fed state all probiotic treatments improved glucose insulinsecretion.

These results are of significance, as low levels of insulin (ie baselevels of insulin) are observed in the fasted state of healthy,non-diabetic subjects. Statistically significant results were achievedfor B420 alone.

Of further significance is that high levels of insulin are observed inthe fed state of healthy, nondiabetic subjects. Statisticallysignificant results were achieved for B420 alone, NCFM alone and thecombination of the two.

Insulin Sensitivity

FIG. 12 illustrates that high-fat diet fed mice were clamped inhyperinsulinemic euglycemic condition by the clamp method. The data showthat B420 treated mice where characterized by a normalization of insulinsensitivity.* p<0.05 vs HFD mice. However, in the presence of NCFM thiseffect was not observed.

These results are of significance, as insulin sensitivity provides thelink between insulin behaviour and consumption of glucose. The resultsshown with B420 are of particular interest since, in comparison withclassic anti-diabetic drugs which target only fasted insulin, insulinsensitivity, or fed insulin, it has an effect on all these factors.

Liver Tissue Inflammation

When considering all cytokine mRNA concentrations, HFD inducedinflammation in liver tissues (FIGS. 13 and 14). Probiotic treatment hadclear anti-inflammatory effect on the liver tissue. This wasparticularly evident with NCFM treatment. Also treatment with thecombination of NCFM and B420 reduced in reduction of inflammation, whileB420 treatment alone reduced inflammation to lesser extent.

Muscle Tissue Inflammation

Inflammation was induced by high fat diet also in muscle tissues,although the induction of inflammation was not as strong as in adiposetissue (FIGS. 15 and 16). Probiotic treatment with B420+NCFM and NCFMalone tended to lower muscle tissue inflammation, but the effect was notas clear as with adipose tissue or liver tissue.

Adipose Tissue Inflammation

The high fat diet clearly induced inflammation in subcutaneous adiposetissue (FIGS. 17 and 18). The B420 treatment and the B420+NCFM treatmentboth showed strong anti-inflammatory effect. Treatment with NCFMresulted in more inconsistent effects on tissue inflammation but therewas a general trend for reduced inflammation.

Taken together, probiotic bacteria showed broad anti-inflammatoryeffect, with most pronounced effects in adipose tissue and liver tissue.It is notable that the anti-inflammatory effects were differential anddependent on the tissue as well as the probiotic treatment.

Example 2

Materials and Methods

A cohort of C57Bl/6 10-wk-old male mice were a high-fat diet (HFD)(comprising 72% fat (corn oil and lard), 28% protein and <1%carbohydrates) (SAFE, Augy, France) for 4 weeks as described inExample 1. The mice underwent an intraperitoneal glucose tolerance test.The area under curve was calculated and the mice dispatchedhomogeneously according to the different experimental groups or ten miceper group (10 mice per group). The mice were fed four more weeks withHFD. The HFD mice were treated daily for 4 weeks as follows with B420(10⁹ bacteria per mouse), polydextrose (FDX) (0.2 g/day), theantidiabetic drug metformin (MET) (2 mg/mL drinking water), and variouscombinations of these. Control mice were treated with saline. Mice werehoused in a controlled environment (inverted 12-h daylight cycle, lightoff at 10:00 a.m.). Blood glucose, insulin concentration and HOMA-IRwere measured from plasma in fasted state.

Results

Treatment either with B420 alone or the combination of B420 andpolydextrose reduced fasting plasma glucose as compared to control.Metformin alone did not have effect on fasting blood glucose but acombination with metformin and polydextrose was effective (FIG. 19).

Treatment with B420 reduced fasting plasma insulin. Addition ofpolydextrose further improved the effect, suggesting a synergisticeffect of the combination. Metformin reduced the fasting plasma insulin,but addition of B420 together with metformin further improved the effect(FIG. 20).

Treatment with B420 reduced fasting HOMA-IR. Addition of polydextrosewith B420 further improved the effect, suggesting a synergistic effectof the combination. Addition of metformin to B420 or B420+polydextrosefurther improved the effect (FIG. 21).

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the following claims.

1-44. (canceled)
 45. A method of treating a disease or condition in amammal, wherein: the method comprises administering to a mammal in needof such treatment a combination of (i) a bacterium of the genusBifidobacterium and (ii) an antidiabetic drug; and the disease orcondition is selected from: (a) diabetes; (b) metabolic syndrome; (c)impaired glucose tolerance; (d) reduced insulin sensitivity; (e) reducedfed insulin secretion; (f) elevated fasted insulin secretion; (g)obesity; (h) weight gain; (i) elevated body fat mass; and (j) elevatedmesenteric fat mass.
 46. The method of claim 45, wherein: the methodcomprises treating diabetes, and the diabetes is Type 2 diabetes. 47-51.(canceled)
 52. The method of claim 45, wherein the mammal in need of thetreatment ingests a high-fat diet.
 53. The method of claim 45, whereinthe Bifidobacterium is a probiotic Bifidobacterium.
 54. The method ofclaim 45, wherein the Bifidobacterium is selected from the speciesBifidobacterium lactis, Bifidobacterium bifidium, Bifidobacteriumlongum, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacteriuminfantis, Bifidobacterium catenulatum, Bifidobacteriumpseudocatenulatum, Bifidobacterium adolescentis, and Bifidobacteriumangulatum.
 55. The method of claim 54, wherein the Bifidobacterium is ofthe species Bifidobacterium animalis.
 56. The method of claim 55,wherein the Bifidobacterium is of the species Bifidobacterium animalissubsp. lactis.
 57. The method of 45, wherein the Bifidobacterium is ofthe species Bifidobacterium animalis subsp. lactis strain 420 (B420).58. The method of claim 45, wherein the Bifidobacterium and antidiabeticdrug are administered in combination with an additional bacterium of thegenus Lactobacillus.
 59. The method of claim 58, wherein the additionalbacterium is of the species Lactobacillus acidophilus.
 60. The method ofclaim 59, wherein the additional bacterium is Lactobacillus acidophilusstrain NCFM (ATCC PTA-4797).
 61. The method of claim 45, wherein theBifidobacterium and antidiabetic drug are administered in combinationwith a prebiotic.
 62. The method of claim 61, wherein the prebiotic ispolydextrose.
 63. (canceled)
 64. The method of claim 45, wherein theantidiabetic drug is selected from a biguanide, a sulfonylurea, analpha-glucosidase inhibitor, a thiazolidinedione, a meglitinide, adipeptidyl peptidase-4 (DPP-4) inhibitor, a glucagon-like peptide-1analog, an amylin analog, a fast acting insulin analog, a long actinginsulin analog, a dual PPAR agonist and an SGLT2 inhibitor.
 65. Themethod of claim 45, wherein the antidiabetic drug is a biguanide. 66.The method of claim 45, wherein the antidiabetic drug is metformin. 67.The method of claim 45, wherein the bacterium and antidiabetic drug areadministered to the mammal as part of a food product, feed product,dietary supplement or medicament.
 68. The method of claim 45, wherein amixture of at least two Bifidobacterium strains are administered to themammal.
 69. The method of claim 45, wherein at least two of the diseasesor conditions (a)-(j) are treated.
 70. The method of claim 45, whereinthe antidiabetic drug is selected from metformin, carbutamide,chlorpropamide, glibenclamide, gliclazide, glimepiride, glipizide,gliquidone, tolazamide, tolbutamide), acarbose, miglitol, voglibose,pioglitazone, rivoglitazone, rosiglitazone, nateglinide, repaglinide,mitiglinide, alogliptin, saxagliptin, sitagliptin, vildagliptin,exenatide, liraglutide, albiglutide, pramlintide, insulin lispro,insulin aspart, insulin glulisine, insulin glargine, insulin detemir,aleglitazar, dapagliflozin, remogliflozin and sergliflozin.
 71. Themethod of claim 70, wherein the antidiabetic drug is selected fromalogliptin, saxagliptin, sitagliptin and vildagliptin.
 72. The method ofclaim 64, wherein the antidiabetic drug is a dipeptidyl peptidase-4(DPP-4) inhibitor.